MicroRNAs (miRNAs) have emerged as powerful regulators of the genome and, through concerted efforts to identify their function and evaluate their ability to alter cell growth in vitro and in vivo, some have gained favor as potential therapeutics. Although these miRNA-based approaches can revolutionize the way that tumors are diagnosed and treated, our understanding of the functional and the molecular aspects of miRNA biology are still incomplete. Moreover, in order to bridge miRNA biology with clinical utility, the challenges that still remain with regard to in vivo delivery of miRNAs must be tackled. To address these challenges we propose two Specific Aims: I. To enhance miRNA therapeutic efficacy through combinatorial miRNA-based targeting and molecular profiling and II. To develop and test second-generation vehicles for delivery of unprotected miRNAs. Our extensive preliminary evidence supports both Aims. We recently identified 10 miRNAs out of 2,019 that significantly enhance the tumor suppressive activity of miR-34a, the first miRNA to enter into clinical trial, and have begun to identify the direct targets of these miRNAs to gain insight into the molecular reason for the cooperative effect. We used a novel method that relies on ligating the cellular miRNAs directly to their associated RNA target followed by deep sequencing of the RNA hybrids. The sequencing data from 13 libraries that we constructed will be used to identify the direct targets of these miRNAs, independent of current algorithms. Targets will be validated and evaluated for pathways that they associate with that will begin to explain their cooperative effect with miR-34a. We also propose to evaluate the in vivo efficacy of the combinatorial pairs using various models of lung cancer. Although we are committed to understanding how these miRNAs are cooperating with miR-34a we also propose to use this data to better understand miRNA biology at a global level. Thus, our data will be used to determine how miRNAs associate with their targets at nucleotide resolution, and how the target population changes with regard to miRNA concentration, which is extremely important to understand as miRNA clinical utility increases. In parallel we will develop and test a second-generation miRNA delivery vehicle, which is a first-in-class method for delivering miRNAs completely unprotected. Following systemic delivery using this method, the miRNA accumulates specifically in the tumor and is efficiently taken up by the tumorigenic cells as indicated by target gene repression with no obvious toxicity. Collectively, the data obtained from this work will validate in vivo efficacy for combinatorial miRNA therapeutics, and for the first time will provide evidence for unprotected miRNA delivery. We will also begin to break down the barriers regarding miRNA target identification that until now has been mostly approached using algorithms that lack critical parameters due to a gap in our understanding of how miRNAs bind their targets.